Heat shock factor 1 protects germ cell proliferation during early ovarian differentiation in medaka

Abstract

The heat shock response is important for the viability of all living organisms. It involves the induction of heat shock proteins whose expression is mainly regulated by heat shock factor 1 (HSF1). Medaka (Oryzias latipes) is a teleost fish with an XX/XY sex determination system. High water temperature (HT) inhibits the female-type proliferation of germ cells and induces the masculinisation of XX medaka in some cases during gonadal sex differentiation. Here, we investigated the roles of HSF1 on the proliferation of germ cells using HSF1 knockout medaka. Loss of HSF1 function under HT completely inhibited the female-type proliferation of germ cells, induced the expression of the anti-Mullerian hormone receptor type 2 (amhr2) and apoptosis-related genes, and suppressed that of the dead end (dnd) and heat shock protein-related genes. Moreover, the loss of HSF1 and AMHR2 function under HT recovered female-type proliferation in germ cells, while loss of HSF1 function under HT induced gonadal somatic cell apoptosis during early sex differentiation. These results strongly suggest that HSF1 under the HT protects the female-type proliferation of germ cells by inhibiting amhr2 expression in gonadal somatic cells. These findings provide new insights into the molecular mechanisms underlying environmental sex determination.

Figure 1

Phenotypes of HSF1 KO medaka. (A) Wild-type (WT) sequences of medaka hsf1 and recognition sites of TALENs (underlined). The sizes of the deletions are shown to the right of each mutated sequence (−, deletions). (B) hsp70-DsRed Tg medaka enabling the monitoring of HSP70 expression by DsRed fluorescence. WT (+/+) and HSF1 homozygous mutated (–/–) embryos heated at 37 °C for 1 h were observed for DsRed fluorescence 1 day after heating. (C,D) hsp70 (C) and hsp27 (D) expression in whole-body fry at 0 dph. Relative expression was calculated based on the value of ef1. n = 4. (E) Fertilisation rate (fertilised eggs over obtained eggs) for 5 days in each of three mating groups: WT males and WT females, KO males and WT females, and WT males and KO females. n = 3. (F) Hatching rate (hatched fry over obtained embryos) in WT (+/+) and KO (–/–) embryos incubated at 26 °C or 33 °C. (G–I) Representative normal (G) and abnormal (H) fry incubated at 33 °C during 0 dpf to 0 dph, and the rate of abnormal fry (abnormal fry over hatched fry) (I). Scale bar, 1 mm. (J) Survival rate in WT (+/+) and KO (−/−) fry incubated at 26 °C or 33 °C during 0 dph to 5 dph. (K) Number of germ cells in WT (+/+) and KO (−/−) fry incubated at 26 °C or 33 °C during 0 dpf to 0 dph. *p < 0.05, **p < 0.01.

Figure 2

Genes with significant (p < 0.05) expression differences between HT-treated WT XX (open column) and HSF1 KO XX medaka (closed column) identified by RNA-seq analysis.

Figure 3

Real-time PCR analysis of HSF1 KO XX medaka. (A–C) hsp70 (A), amhr2 (B), and dnd (C) expression in the gonadal regions of HT-treated WT XX (+/+) and HSF1 KO XX (−/−) fry at 0 dph. Relative expression was calculated based on the value of ef1. **p < 0.01, n = 4.

Figure 4

Phenotypic analysis of AMHR2 KO and HSF/AMHR2 KO medaka. (A) WT sequences of medaka amhr2 and recognition sites of TALENs (underlined). Deletion sizes are shown to the right of each mutated sequence (−, deletions). (B,C) amhr2 (B), and dnd (C) expression in gonadal regions of WT XX (+/+) and AMHR2 KO XX (−/−) fry at 0 dph. Relative expression was calculated based on the value of ef1. n = 5. (D) Mating scheme for HSF1/AMHR2 double KO medaka. (E) Number of germ cells in WT (+/+) and HSF1/AMHR2 KO (−/−) fry at 0 dph after HT treatment. *p < 0.05, **p < 0.01.

Figure 5

Detection of apoptosis in HSF1 KO XX medaka. (A–H) Apoptosis with TUNEL staining (green) and nuclear DAPI staining (blue) in gonadal regions of WT XX (A,E), HT-treated WT XX (B,F), HSF1 KO XX (C,G), and HT-treated HSF1 KO XX (D,H) fry. White dot, gonadal regions; white arrow, apoptosis in gonadal regions. Scale bar, 30 μm. n = 4. (I) Rate of apoptotic germ cells (TUNEL-positive germ cells over total germ cells). (J) Rate of apoptotic gonadal somatic cells (TUNEL-positive gonadal somatic cells over total gonadal somatic cells). (K,L) Number of germ cells (K) or gonadal somatic cells (L) of WT (+/+) and KO (−/−) fry at 0 dph. **p < 0.01.